Viral vectors constitute valuable tools in our gene transfer arsenal. The utility of viral vectors is derived mainly from their unsurpassed ability to deliver genes from the plasma membrane to the nucleus. In vivo experience using experimental animals has confirmed the fact that adeno-associated virus (AAV) vectors yield effective and persistent gene transfer with promise for the treatment of chronic genetic diseases such as cystic fibrosis. To optimize clinical use of gene transfer vectors, variables affecting the dynamics of gene transfer vectors must be characterized and understood. Recent evidence has pointed to intracellular trafficking of AAV vectors as a variable in AAV-mediated gene transfer to airway epithelium. The central hypothesis of this proposal is that the lung tropism of select AAV serotypes is derived, in part, from comparatively efficient intracellular trafficking of the genome to the nucleus in differentiated airway epithelial cells. Cells contain numerous barriers to viral infection including the plasma membrane, a viscous cytosol, and the nuclear envelope. Capitalizing on the experience developed in this laboratory in studies of viral interaction with these intracellutar barriers, we propose to pursue three avenues relevant to the intracellular AAV infection pathway: (1) to conduct a comparative analysis of the ability of different AAV serotypes (including lung-tropic serotypes) to traffic through airway epithelial cells to the nucleus, (2) to determine the extent to which intracellular trafficking of AAV serotypes results from an affinity of the AAV capsid for the microtubule cytoskeleton mediated by the cytoplasmic dynein motor complex, and (3) to evaluate whether proteosomal degradation of the AAV capsid correlates with intracellular trafficking of AAV to the microtubute-organizing center (MTOC). Furthering our knowledge of the mechanism of AAV infection will provide valuable insight that can be used for strategic development ofgene transfer vectors.